PROJECT SUMMARY/ABSTRACT Non-small cell lung cancer (NSCLC) accounts for the majority of lung cancer, which to-date remains the leading cause of cancer death in the U.S. and worldwide. About 25% of NSCLC harbors Kras oncogene activating mutations. Because direct therapeutic targeting of Kras proved challenging, strategies shifted to targeting downstream effector signaling pathways. However, their efficacy and toxicity remain under investigation, and alternative therapeutic approaches are urgently needed. Evidence from pharmacological and cell culture studies point to a role for insulin and insulin-like growth factor-1 (IGF-1) signaling in Kras-driven lung cancer. However, the specific contribution of this pathway to Kras-driven tumor initiation and progression is unclear, and its role in altering lung tumor metabolism is unknown. Most, if not all insulin/IGF-1 signaling in the lungs converges intracellularly onto the adaptor proteins insulin receptor substrates IRS1 and IRS2 prior to diverging to a complex network of downstream signaling effectors, including PI3K/Akt. The forkhead transcription factors Foxo1 and Foxo3 are insulin-regulated targets that are inactivated by Akt, and affect cellular metabolism, survival and proliferation. Foxos are well known for regulating hepatic glucose metabolism by promoting glucose production and suppressing its utilization. However, the potential tumor-suppressing roles of Foxos in Kras-driven cancers have not been investigated. Here, using distinct conditional genetically engineered mouse (GEM) models of Kras-driven lung cancer, the effects of genetic ablation of IRS1 and IRS2 on the initiation, maintenance, and metabolism of lung tumors, as well as the roles of Foxo1 and Foxo3 in mediating these effects, will be investigated. Histopathological and in vivo imaging techniques will be used to assess at timepoints concomitant with, or subsequent to Kras activation, the effect of IRS gene loss on lung tumor latency, tumor burden and survival of these mice and whether additional loss of Foxo1 and Foxo3 genes would reverse such effects. Moreover, the differential activation of signaling pathways and expression of genes that regulate glucose utilization will be assayed for, and mass spectrometry analyses of glucose-derived metabolites will be performed on the tumors upon loss of IRS genes in the presence or absence of the Foxo genes. Cells will also be isolated from the Kras-driven tumors and grown in culture. Approaches similar to the ones described for in vivo tumors will then be performed on the cells in vitro, to identify targets downstream of IRSs and Foxos, that can alter glucose utilization and hence affect lung tumor maintenance. The role of these targets can then be confirmed via knockdown/overexpression studies. In addition, similar studies will be performed in established, Kras-driven, human NSCLC cell lines with stable inducible knockdown of IRS1 and/or IRS2 that can also be grown as subcutaneous xenografts in immune-deficient mice. Results from these studies will reveal novel metabolic Kras-driven lung cancer vulnerabilities that could be exploited therapeutically in NSCLC patients. !